KR20030064742A - Oral delivery of peptide - Google Patents

Abstract

PURPOSE: Provided are a proliposome of peptide drug and an oral formulation containing the proliposome. The oral formulation remarkably enhances bioavailability and stability of the peptide drug. CONSTITUTION: The preparation method comprises the steps of: dissolving a peptide drug and phospholipid in an organic solvent; coating the solution with water soluble chitosan to make proliposome; formulating the proliposome into pharmaceutically acceptable formulation; and coating the formulation with fatty acid or its salt to accelerate absorbance in the intestine.

Description

ORAL DELIVERY OF PEPTIDE}

Peptide drugs are rapidly expanding in the 1990s due to the rapid development of genetic recombination technology and solid-state peptide synthesis technology. More than 100 items are developed and marketed.

However, most formulations of currently developed and marketed peptide drugs are injectables.

1) accompanied by pain in the patient when administered to the patient;

2) There are difficulties in storage such as refrigeration

3) Because of the shortcomings of injectables, such as the need to visit the hospital when a drug is administered to a patient, the development of oral preparations of peptide drugs is urgently required, but until now, the developmental stage is insignificant.

However, when the above difficulties as injections of peptide drugs are overcome with oral dosage forms,

1) the dosage can be formulated in the form most suitable for the patient,

2) The stability of the preparation will be increased, so that the production, sale and consumption, such as distribution and expiration date, will be changed more conveniently and advantageously than the existing formulation,

3) As the market of oral dosage forms preferred by patients is expected to quickly replace the existing drug dosage forms, technical research on the development of oral dosage forms is actively conducted.

In general, technical difficulties for the development of oral formulations of peptidic drugs have been noted in prominent papers (Raymond M. Reilly, Rommel Domingo and Jasbir Sandhu.Drug Delivery Systems, 32, 4, 313-323, 1997: Jeoseph A Fix.Pharmaceutical Research.vol. 13. no.12.1996: Amyn P. Sayani and Yie W. Chien.Critical Reviews in Therapeutic Drug Carrier Systems, 13, 1 & 2, 85-184, 1996: Jane PF Bai, Li- Ling Chang and Jian-Hwa Guo, Critical Reviews in Therapeutic Drug Carrier Systems, 12, 4, 339-371, 1995)

Problems to be solved in the development of oral formulations of such peptide drugs

1) low absorption rate in the intestinal tract,

2) loss of potency by small intestine protease,

3) It is the focus of research on the problems that need to be solved due to high half-life in the body after absorption.

One of the ways to solve these problems is to solve the low absorption rate in the intestine.

1) Encapsulating the drug in nano or microsphere particles to increase biofilm passage (Istvan Toth, International Journal of Pharmaceutical, 183, 1999, 51-55: E. Bjork, U. lsaksson, P. Edman. J. Drug Targeting 2, 501-507, 1995: Shinji Sakuma.Norio Suzuki.International Journal of Pharmaceutical.149.1997.93-106),

2) Method of using absorption accelerators together (Patrick J. Shinko, Yong-Hee Lee, Pharmaceutical Research, vol, 16, No 4, 1999, 527-533: JC Scott-Moncrieff, Z. Shao, J. Pharm. Sci 83, 1465-1469, 1994: EA.Hosny, N. M Khan Drug Devel.Ind. Pharm. 21, 1583-1589, 1995: Isao Sasaki, Hideyuki Tozaki, Biol. Pharm. Bull. 22, 6, 611- 615, 1999: Anthomy C. Chao, Joseph Vu Nguyen, International Journal of Pharmaceutical, 191, 1999, 15-24),

3) Method of modifying peptide structure (Deven Shah, Wei Chiang, Journal of Pharmaceutical Sciences, vol 85, No 12, 1996, 1306-1309: Kazunori Iwanaga, Satoshi Ono, Journal of Pharmaceutical Sciences, vol 88, No 2, 1999 , 248-252: Istvan, John P. Malkinson, J. Med. Chem. 1999, 42, 4010-4013)

4) Method of altering the structure of the drug or using an enzyme inhibitor together (P. Buhlnayer, A. Caseli. W. Fuhrer. J. Med. Chem. 31, 1839, 1998: A. Yamamato. T. Taniguchi, K Rikyu, T. Tsuji, Pharm.Res. 11, 1496-1500, 1994)

5) Incorporation into microparticles to increase stability in the body after absorption, formulas of drugs (David F. Ranney, Biochemical Pharmacology, vol. 59, 105-114, 2000: Ian M. Chapman, Ora H. Pescovitz, Gail Murphy, Journal of clinical Endocrinology & Metabolism.1997. Vol 82, No 10, 3455-3463) has been used mainly, and research on the development of formulations for peptide drugs has been actively conducted in various ways (Z. Aydin, J. Akbuga.International Journal of Pharmaceutics 131. 101-103, 1996: K. Aledeh, E. Gianasi, I. Orienti and V. Zecchi.J. Microcapsulation, 1977, vol. 14, no. 5, 567-576: A. Pork.B. Amsden.K. De yao Journal of Pharmaceutical Science vol. 83. No. 2, 1994. 178-185).

Meanwhile, the method using liposomes as a method of developing oral formulations of these peptide drugs

1) It is easy to encapsulate drugs in relatively fine particles (nano or microsphere particles).

2) Liposomes can be easily absorbed in the mesenteric membrane, and many studies have been conducted in recent years, and prior arts about them include a technology disclosed by Lipotec, SA (EP- 0855179 A2).

However, this method is useful for preparing liposomes.

1) Because of using water, there is a problem with the stability of the drug,

2) there is a problem such as using a process that undergoes a freeze-drying or drying process to produce a powder form

3) The stability in aqueous solution is low due to the liposomes' physical properties such as aggregation, sedimentation, fusion, oxidation and phospholipid hydrolysis of liposomes themselves.

4) There were disadvantages to use as medicine because of difficulty in reproducibility and sterilization in production process.

The present invention relates to oral dosage forms of peptides and protein medicines (hereinafter referred to as "peptide drugs") and a method for producing the same.

1 is a photograph showing the formation of proliposomes and internal encapsulation of drugs with water-soluble chitosan of salmon calcitonin,

Figure 2 is a photograph showing the process of dissolving in water by observing the hydration process of the proliposomes using a water-soluble chitosan, Figure 2a is a photograph showing a proliposomal powder, Figure 2b shows that liposomes are produced in the proliposomes It is a photograph.

Figure 3 is a graph showing the particle size and distribution of liposomes formed by the hydration of proliposomes.

Figure 4 is a graph showing the change over time content of salmon calcitonin in the proliposomes of salmon calcitonin (salmon calcitonin) according to the present invention.

Figure 5 is a graph showing the drug transmittance of various formulations containing salmon calcitonin-containing proliposomes as an embodiment according to the present invention.

The present inventors have completed the present invention as a result of a long time conducting a pharmaceutical research review focusing on the physical formula rather than the chemical formula of the peptide drug to overcome the problems of the prior art.

The present invention provides a method for improving the problems with these prior arts.

1) Proliposome, a precursor of liposomes, was prepared using water-soluble chitosan to increase absorption in the intestinal membrane.

2) using a pH adjuster to increase the stability of the peptide drug in the intestinal aqueous solution,

3) After mixing the additives such as absorption accelerator so that the peptide drug can be easily absorbed from the membrane

4) At the same time it is formulated into a formulation suitable for oral administration,

5) It is a method of preparing the formulation so that it can be easily absorbed and moved to the intestine without breaking down the peptide drug in gastric acid by enteric coating.

In other words, the present invention provides a high yield in a short time to the proliposome, which is a preliminary step of liposomes, without undergoing a process such as lyophilization or evaporation to prepare liposomes in powder form, which is a problem in the conventional method for preparing oral preparations using liposomes, such as EP-0855179. It can be prepared as a simple production process, and has the advantage of increasing the bioavailability by using the chitosan as a carrier for producing proliposomes, increasing the stability to moisture and temperature, which is a disadvantage of the peptide drug .

Specific contents of the present invention,

1) Using the water-soluble property of water-soluble chitosan, a peptide-type drug coated with water-soluble chitosan in powder form using an organic solvent was prepared in powder form,

2) after enteric coating to protect the peptide drug from stomach acid,

3) The physical properties of water-soluble chitosan dissolved in water when the formulation reaches the intestine allows the proliposomes in powder form to form liposomes in the intestine so that they can be absorbed smoothly in the intestinal membrane during oral administration. The nature of membrane adhesion,

4) In order to prevent most peptide-based drugs from being unstable in aqueous solution and easily decomposed in the intestine after oral administration, most of the peptide-based drugs are stable at pH 3-4 to increase intestinal stability. Using,

5) Peptide drugs can be easily absorbed in the mesentery membrane by mixing and mixing additives such as absorption accelerators so that the peptide drug in the mesentery membrane is easily absorbed into the paratrans patten between cells.

Next, the content of the present invention will be described in detail.

Chitosan is a natural polymer made from polysaccharide kitchens that support and protect the living organisms widely distributed in shells of crustaceans such as crabs and shrimps, epidermis of insects, cell walls of mushrooms and fungi.

In other words, kitchen is a cellulose-like structure that is a very long chain, high-molecular-molecular substance, in which acetylamino group (-NHCOCH ₃ ) is substituted for carbon number 2 instead of hydroxyl group (-OH), and chitosan is carbon number 2 in kitchen. It is a substance obtained by deacetylating an acetyl group linked to an amino group, and has various physiological activities because it can have many free cations.

For this reason, high quality chitosan has been developed and used in various fields such as food, cosmetics, medicines and adsorbents, activators of plant cells, and wastewater treatment flocculants.

Especially, in recent years, the fat binding ability of chitosan is much more effective than vegetable fiber, and it is shown to be harmless because it not only improves immunity and resistance to disease but also has no toxicity in vivo.

However, chitosan, which has been used for all of you, is not soluble in water or alcohol, but is only dissolved in organic acid solutions such as formic acid, lactic acid and acetic acid, and inorganic acids such as dilute hydrochloric acid.

Therefore, in the present invention, the oral administration of the peptide drug using chitosan (JK FM-01: Gwangkwang), which is soluble in water, has completed the present invention, and the water-soluble chitosan used in the present invention

1) Deacetylation degree 85%-99%

2) Solubility in water 99.99% or more

3) Molecular weight is 100,000-500,000 and can be used as excipient in medicine

4) It has the feature of conforming to food additive standard.

In other words, the chitosan used in the present invention shows that 99.99% or more is dissolved in water as its main characteristic.

1) After deacetylation, which is a method of hydrolyzing an acetylamino group by hydrolyzing an aqueous solution of chitin, a chitosan production method, to make an amino group, it is water-soluble and deacetylation degree is 85% Increased to 99%,

2) The purity of chitosan was manufactured to more than 90% through a multi-stage membrane process,

3) The amino group was activated during the deacetylation of the second carbon of chitosan, so it easily dissolves in water while having the characteristics of chitosan when contacted with water molecules.

In addition, the feature of the present invention solves the problems of liposomes that have been used in the field of medicines and cosmetics to date, while at the same time retaining the advantages of chitosan and using the advantages of water-soluble chitosan dissolved in an aqueous solution of the peptide-based drug To manufacture.

Liposomes are monolayer or multilayered phospholipid bilayer vesicles (Bangham et al, Diffusion of Univalent Ions across the Lamellae of Swollen Phospholipids, J. Mol. Biol., 13, 238-252. 1965 Liposome itself according to the manufacturing method since

1) surface charge,

2) size,

3) It is possible to modify lipid content, etc., and has been produced by various manufacturing methods for several decades according to the intended purpose and use, and has been used in food and cosmetic medicine.

Especially in medicine

1) sustaining the drug or targeting the drug

2) alleviation of acute toxicity and alleviation or enhancement of immune response

3) stabilization of the drug

4) It is used to develop various formulations of medicines for the purpose of changing dosage forms.

However, liposomes that have been used to develop pharmaceutical formulations

1) cohesiveness of liposomes,

2) sedimentation,

3) fusion;

4) oxidation,

5) low stability in aqueous solution due to the liposome itself properties such as hydrolysis of phospholipids,

6) In the mass production process, the sterilization and reproducibility efficiency was low, so it was insufficient to be used efficiently in medicine.

For this reason, the present inventors have studied for a long time to solve the problems of the conventional liposome preparation, and when the proliposome in the form of a solid powder, which is a preliminary step of the liposome with a water-soluble chitosan as a carrier, the problem of the conventional liposome preparation The present invention has been completed by discovering surprising facts that all solve.

Accordingly, it is an object of the present invention to provide proliposomes of peptidic drugs prepared using water soluble chitosan.

Another object of the present invention is to provide an oral preparation in which a proliposome prepared by using a water-soluble chitosan with a peptidic drug is formulated by a conventional pharmaceutical method in a conventional manner.

It is still another object of the present invention to provide a preparation method for preparing an oral preparation in which a proliposome prepared by using a water-soluble chitosan for a peptide drug is prepared by a conventional pharmaceutical method.

In general, the characteristics of the carrier for preparing proliposomes

1) It must be water-soluble because it must be dissolved in water and the proliposome must be transformed into liposome.

2) It should be characterized by low solubility in organic solvents used in the manufacturing process.

The water-soluble chitosan used in the present invention not only has the carrier property of such proliposomes, but also when dissolved by water in the intestine, when the oral administration of the peptide drug by the thickening effect due to the fat-binding ability of the free cation possessed by chitosan itself. Can increase the bioabsorption rate of the drug.

Peptide drugs that can be used in the present invention include aprotinin, buserelin, calcitonin, desmopressin, elcatonin, glucagon, gonadotro Gonadotropin, gonadorelin, goserelin, hirudin, leuprorelin, lypressin, nafarlin, octreotide , Oxytocin, protirelin, salcatonin, sercalinin, sermorelin, somatostatin, somatotropin, terrilpressin, terlipressin, tetracosacrine tetracosacrin, thymopentin, triptorelin, triposolin, vasopressin, albumin, insulin, interferon, immunoglobulin, GM-CSF, G- CSF, glycoproteins (glycoprotein) and the like. In addition, peptides and protein drugs are also included in the scope of the present invention.

Phospholipids used in the preparation of peptidic drug-containing proliposomes of the present invention include pharmaceutically acceptable conventional phospholipids used in the preparation of liposomes, for example, phosphatidyl-diesel-glycerol-dimyristoyl (Phosphatidyl- DL-Glycerol-Dimyristoyl), L-α Egg phosphatidyl choline, Soybean phosphatidyl choline (lecithin), Dipalmitoyl phosphatidyl choline (DPPC) ), Dimyristoyl phosphatidyl choline (DMPC), cholesterol (Cholesterol), stearylamine, diacetylphosphate, phosphatidyl serine, methoxy polyethylene glycol distearo One phosphatidyl-ethanolamine (Methoxy polyethylene glycol distearoyl phosphatidyl-ethanolamine) and the like can be used.

Another feature of the present invention is that the peptide drug, which is generally composed of amino acids, contains asparagine or glutamine residues in its structural components, and these residues are deamidation and beta-yl in aqueous solution. Reactions such as beta-elimination, disulfide exchange, racemization, oxidation, etc., resulting in easy structural modification and ultimately loss of the activity of the peptide drug. When the oral dosage form according to the present invention is dissolved in the intestine, asparagine or glutamine do not cause a chemical reaction by adjusting the pH 3-4 range using a pH adjuster to maintain the peptide drug in the intestine.

In general, the pH adjusting agent used in the present invention can be used for oral use in medicine. Representatives include citric acid, sodium citrate, adipic acid, sodium dihydrogen phosphate, etc. desirable

Another feature of the present invention is to promote absorption of the drug in the small intestine upon oral administration of the peptidic drug using a natural or food-derived absorption promoter that does not damage the intestinal epithelial cells upon intestinal absorption. Absorption accelerators that can be used in the present invention are fatty acids or salts thereof, such as capric acid, oleic acid and the like, and salts of bile acid-based cholate and deoxycholic acid ( deoxycholic acid) and salicylate (salicylate), such as the absorption promoter of the mologlyceride (monoglyceride) system, etc. can be used. The absorption promoter mainly used in the present invention in the embodiment of the bile acid system It is preferable to use the deoxycholine acid salt which is a salt, and it is preferable to use 0.1%-10% of usage.

Next, the present invention will be described in detail by Examples and Experimental Examples. However, the scope of the present invention is not limited to this embodiment and the experimental example.

Example 1

Preparation of Proliposome (Preparation of Salmon Calcitonin)

4 g of water-soluble chitosan powder (106 µm-300 µm) in a sieve was placed in a 100 ml round bottemed flask, placed in a rotary evaporator, and dried under reduced pressure at room temperature for 30 minutes. As phospholipid, 267 mg of egg lecithin was dissolved in 30 ml of chloroform, and then mixed with a solution of 27 mg of salmon calcitonin dissolved in 10 ml of ethanol in advance.

While maintaining the temperature in a 100 ml round bottom flask containing water-soluble chitosan at 20-30 ° C, add a certain amount (about 1 ml) of the organic solvent mixture in which liposome components are dissolved, and rotate the rotary evaporator at 120-150 rpm. While maintaining the water-soluble chitosan completely dried and free-flow (free-flow) state, the organic solvent mixture was added again and repeatedly coated. The final solution is added, dried and re-dried in a freeze dryer for 24 hours to remove the organic solvent (Yield: 95% or more).

Example 2

Preparation of Proliposomes (Preparation Example of Desmopressin)

3 g of a water-soluble chitosan powder (106 µm-300 µm) in a sieve was placed in a 100 ml round bottom flask, placed in a rotary evaporator, and dried under reduced pressure at room temperature for 30 minutes. As a phospholipid, 160 mg of phosphatidyl-DL-glycerol-dimyristoyl was dissolved in 24 ml of chloroform and mixed with a solution of 20 mg of desmopressin previously dissolved in 12 ml of ethanol.

Maintain the temperature in a 100 ml round bottom flask containing water-soluble chitosan at 20-30 ℃ and add a certain amount (about 1 ml) of the organic solvent mixture in which liposome components are dissolved, and maintain the rotational speed of the rotary evaporator at 120-150 rpm. While the water-soluble chitosan is completely dried and free-flowing state, the organic solvent mixture is added again and repeatedly coated.

The final solution is added, dried and re-dried in a freeze dryer for 24 hours to remove the organic solvent (Yield: more than 96%)

Examples 3-33

Aprotinin, Buserelin, Elkatonin, Glucagon, Gonadotropin, Gonadorelin, Goserelin, Hirudine, Leproproline, Ripressin, Nafarerlin, Octreotide, Oxytocin, Protyreline, Salkatto Nin, Sermorelin, Somatostatin, Somatropin, Terylpressin, Tetracosacrine, Tymopentin, Tryptorelin, Vasopressin, Albumin, Insulin, Interferon, Immunoglobulin, GM-CSF, G-CSF, Glyco Each peptide-containing drug-containing proliposome was prepared in a manner similar to that of Example 1 or 2, using proteins of protein, respectively.

Experimental Example 1

Confirmation of drug loading of proliposomes (Cryo -SEM confirmation)

In order to confirm the drug encapsulation of the proliposome containing salmon calcitonin, it was confirmed that the drug was encapsulated in the proliposome by comparing the proliposome without the drug with the proliposome with the drug using a cryogenic electron microscope. First, drop a liquid sample (liposomes hydrated proliposomes) on a 1 cm diameter disc bed (approximately 3 μl) and use a cryo-transfer system (CT 1500 Cryotrans, Oxford Instrument Ltd., UK). Fill the liquid nitrogen with the nitrogen slushing chamber of) and rapidly put the sample into it, and fix it for 1 minute under vacuum.

The sample was then transferred to a cryo-chamber controlled at -170 ° C., checked for vacuum, and randomly crushed and cut the cooled sample, and the crushed sample was connected to a cryo-transfer system. JSM-5410LV, JEOL LTD., Japan) is moved to the sample stage, the temperature is adjusted to -70 ℃ and maintained for 5 minutes to sublimate the surface moisture.

After the sublimation process, it was continuously moved back to the cryo chamber and coated with gold (gold-coating), and then the fractured and cut surface of the sample was observed by scanning electron microscope (SEM) at an acceleration voltage of 20 kV. The formation of liposomes and the internal encapsulation of the drug were confirmed and the photograph is shown in FIG.

Experimental Example 2

Prolysis of proliposomes:

In order to observe the hydration process of proliposomes using water-soluble chitosan under an optical microscope, the proliposomal granules were placed on a microscope slide, focused, and magnified at 400 times to drop a drop of water onto the granules. The process of dissolving proliposomes in water was confirmed and this photo is shown in FIG. 2.

Experimental Example 3

Particle size analysis of manufactured liposomes:

To 5 mg of the water-soluble chitosan proliposomes prepared in Example 1, 5 ml of distilled water was added, shaken while vortexed, and hydrated for 30 minutes. The particle size and distribution of the liposomes thus formed were measured using a particle size analyzer. The results are shown in the graph of FIG.

Experimental Example 4

Determination and Stability of Salmon Calcitonin in Proliposomes:

To confirm stability and quantification, the prepared proliposomes of about 5 ㎍ of salmon calcitonin are precisely taken, dissolved in 300 μl of methanol, and vortexed to completely dissolve. After filtering to 0.45㎛ 40μL injected into HPLC to quantify salmon calcitonin in proliposomes and the results for stability at room temperature and refrigeration is shown in Figure 4, the conditions of HPLC used for drug content analysis Same as

HPLC conditions: Column-C18 (sucelpo)

Flow rate-1 ml / min

Solvent A-Acetonitrile (0.1% TFA)

Solvent B-Water (0.1% TFA)

Experimental Example 5

Formulations for Oral Administration (Examples of Tablets) and Enteric Coatings:

Tablets were prepared by direct method using dry method instead of wet method according to general tablet manufacturing method of the general pharmacopeia formulation part.The prescription of each sample prepared to verify the effect of the invention is shown in Table 1, and each sample manufactured by direct method Enteric coating was coated using CAP, and the coating results were confirmed by disintegration tests in 1 and 2 solutions using the Pharmacopoeia General Test Method. This result is shown in Table 2.

division Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 1 liquid Not recording Not recording Not recording Not recording Not recording 2 liquid record record record record record

In vivo and in vitro experiments were run together to verify the effect of the invention.

Experimental Example 6

In vitro experiments with cells:

(1) Complete Media Manufacturing

Add 2 liters of DMEM (dulbecco's mocifued eagle medium), room temperature 7.4 g sodium bicarbonate and 2.6 g HEPES in 2 liters of sterilized and cooled distilled water at room temperature for about 1 hour-1 hour 30 minutes in a plate stirrer. Stir.

After stirring, the solution was adjusted to pH 7.4 with 1N hydrochloric acid using pH-meta. The solution was filtered using a filter (corning filter 430015), and then dispensed into a 500 ml bottle using a 50 ml pipette.

Put 50ml of FBS, 5ml of streptomycin, and 5ml of NEAA in 500ml bottles dispensed in 450ml each. To check whether the prepared medium is contaminated, put about 6ml of complete medium in a 25ml T-flask and observe it under a microscope. Check for contamination after incubation for more than 1 day.

(2) defrosting of CaCo ₂ cells

The complete medium, which was prepared and stored in a refrigerator, was warmed for 20 minutes in a 37 ° C water bath, and the contents were mixed 4-5 times using a 5ml pipette, and then 5ml was put in a 15ml conical tube and a nitrogen tank. Remove the cell line (ATCC HTB-37, LOT 944495) from the (Nitro tank), open the bottle cap slightly, dissolve in a 37 ° C water bath, confirm that it is completely dissolved, and open the lid of the cell line storage bottle. Mix 5 to 6 times and add to the 15 ml conical tube.

Centrifuge the 15ml conical tube at 1000rpm for 7 minutes, take out the supernatant, add 1ml of media, mix with a pipette 4-5 times to release the cells, and then remove the solution of the 15ml conical tube. The entire volume is placed in a 25 ml T-flask and the 25 ml T-flask is stored in an incubator.

(3) Feeding cell media change

Remove the 25 ml T-flask stored in the incubator, remove the medium from the bottom of the flask, warm the complete medium, which has already been prepared and stored in the refrigerator, in a 37 ° C water bath for 20 minutes, take 5 ml, and 25 ml T-flask Replace the medium taking care not to touch the pipette

(4) Subculture

The trypsin EDTA container, free media, and complete medium stored in the refrigerator were warmed for 20 minutes in a 37 ° C water bath, then the medium of the 25 ml T-flask in the incubator was removed and the trypsin EDTA was placed in the 25 ml T-flask. Put 2ml.

Trypsin EDTA is spread evenly and stored for 4 minutes in a 37 ℃ water bath, 10ml of complete medium in a 25ml T-flask, shaking the flask to separate the cells, transfer the separated solution to a 15ml conical tube using a pipette and 5 at 1000rpm After centrifugation for a minute, the supernatant is removed, 2 ml of complete medium is taken into a T-flask, the cells are allowed to dissipate well and re-incubated in an incubator (in this experiment, more than 40 passages were used).

(5) Coating of Transwell Membrane with Collagen

10 ml of sterile 0.1% acetic acid was added to 25 mg of rat tail collagen, and then the bar magnet was added and stirred for 3 to 5 hours to dissolve. The solution was diluted to 1: 1.5 using 60% ethanol solution and the final concentration 0.3 mg / ml, and then 50 ml of collagen solution is spread evenly over the upper membrane of the transwell (Costar 3401), the lid is opened and the UV light source is turned on in the laminar flow and left for 4-5 hours. To dry.

(6) Seeding cells onto Transwell plate

Put 0.5ml of complete medium in the upper part of the coated transwell and 1.5ml in the lower part, incubate in the incubator for 15 minutes, remove the medium and put 1.5ml of complete medium in the lower part of the coated transwell again. After subculture, dilute cells of more than 40 passages to a concentration of 2.5-3 x 10 ⁵ cells / ml, add 1.5 ml to the top of the coated transwell, and use them for cell permeation experiments for 2 weeks. Incubate for 3 weeks.

(7) Identification of CaCo ₂ cell monolayer

Measurement of Transepithelial Eledtrical Resistance (TEE) was measured at least 250 kW 2 cm after 2-3 weeks after inoculation into the transwell using a Millicell-ERS Resistance System. It was confirmed that the monolayer film of the cell was formed well, and the permeation experiment using C ¹⁴ -mannitol (<receiver dpm> / <donor dpm>) / hr / ㎠ showed that the monolayer film was well formed. The specific details of the experiment were as follows.

Drug concentration used: C ¹⁴ -Mannitol 2-10μM with specific activity 50 mCi / mmol

Quantification of C ¹⁴ -mannitol: 100 μl each was sampled into an LSC vial, 2 ml of LSC cocktail was added, and vortexed for 10 seconds to measure dpm using a liquid scintilation counter (LSC).

(8) Drug Permeation Experiment (Example of Salcatonin)

Remove the top medium of the verified transwell using a pipette, dissolve the proliposome prepared in Example 1 in the transbuffer at a concentration of drug (4.0 ㎍ / ml), adjust the concentration, and place it on the top of the transwell. Shake water slowly at 85 rpm using a shaking water bath at 37 ° C and transfer the transwell to a new well containing a flashbuffer every 15 minutes after drug administration.

Drugs were sampled from the wells of the donor section of the transwell and quantified by salivatonin using an ELISA and RIA Kit (Pen. Lab). The result is shown in FIG.

Experimental Example 7

In vivo experiments using rats

250g-300g SD rats (5 dogs per group) were fed only 1 day prior to the start of the experiment and adapted to the experimental environment.Then, anesthetics Ketamine: Rumfu was used to insert cannula into the femoral vein and artery. : Inject the mixture at the ratio of 4 and inject the muscle with 400 μl. Then, fix the rat on the operating table and find the femoral vein and artery of the leg using scissors and tweezers.

After inserting the cannula, the blood was drawn from the veins and arteries with a syringe to confirm the blood collection. Then, oral capsule sonde was used to orally administer a sample of Formulation 1, Formula 2, Formula 3, Formula 4, or Formula 5 to the rat. Collect blood samples from the femoral arteries at regular time intervals by 500 μl and injecting the corresponding amount of saline into the femoral vein.

The collected blood is centrifuged at 10,000 rpm for 10 minutes in a centrifuge, and then the supernatant is collected separately and the drug is quantified using Elisa and RIA Kit (Pen. Lab).

Separately, to compare the increase in bioavailability, the same amount of orally administered drug was injected into the femoral vein, and pretreatment was performed in the same manner as the blood collection method in the formulation. .

The effect of the present invention,

1) Proliposomes, which are precursors of liposomes, were prepared using water-soluble chitosan to increase absorption in the intestinal membrane.

2) using a pH adjuster to increase the stability of the peptide drug in the intestinal aqueous solution,

3) After mixing the additives such as absorption accelerator so that the peptide drug can be easily absorbed from the membrane

4) At the same time it is formulated into a formulation suitable for oral administration,

5) It is a preparation method of preparing the formulation so that it can be easily absorbed and moved to the intestine without breaking down the peptide drug in gastric acid.

In other words, the present invention provides a high yield in a short time to the proliposome, which is a preliminary step of liposomes, without undergoing a process such as lyophilization or evaporation to prepare liposomes in powder form, which is a problem in the conventional method for preparing oral preparations using liposomes, such as EP-0855179. It can be prepared as a simple production process, and has the advantages of increasing the bioavailability by increasing the stability against moisture and temperature, which is a disadvantage of the peptide drug, and by using a water-soluble chitosan as a carrier for producing a proliposome have.

Claims (8)

Proliposome prepared by dissolving peptide drug and phospholipid in organic solvent and coating this solution with water-soluble chitosan. The method of claim 1, wherein the peptidic drugs include aprotinin, buserelin, calcitonin, desmopressin, elkatonin, glucagon, gonadotropin, gonadorelin, goserelin, hirudin, leuprorelin, and ripressin , Naparerine, Octreotide, Oxytocin, Protyreline, Salcatonin, Sermorelin, Somatostatin, Somatropin, Terylpressin, Tetracosacrine, Tymopentin, Tryptorelin, Vasopressin, Albumin, Insulin Proliposomes, characterized in that selected from interferon, immunoglobulin, GM-CSF, G-CSF and glycoprotein. The proliposome according to claim 1 or 2, wherein said water-soluble chitosan has a deacetylation degree of 85%-99% and a molecular weight of 100,000-500,000. The method according to claim 1 or 2, wherein L-alpha egg phosphatidyl choline, soybean phosphatidyl choline, dipalmitoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, cholesterol, stearylamine, diacetylphosphate, phosphatidyl serine and methoxy A proliposome characterized by using a phospholipid selected from polyethylene glycol distearoyl phosphatidyl ethanolamine. Proliposomes prepared by dissolving peptide drugs and phospholipids in organic solvents and coating the solution with water-soluble chitosan are formulated in the usual pharmaceutically acceptable formulations in the form of conventional pharmaceutically acceptable formulations. An enteric preparation of proliposome of a peptide drug prepared by enteric coating by a method. The method of claim 5, wherein the peptidic drugs are aprotinin, buserelin, calcitonin, desmopressin, elkatonin, glucagon, gonadotropin, gonadorelin, goserelin, hirudin, leuprorelin, ripressin , Naparerine, Octreotide, Oxytocin, Protyreline, Salcatonin, Sermorelin, Somatostatin, Somatropin, Terylpressin, Tetracosacrine, Tymopentin, Tryptorelin, Vasopressin, Albumin, Insulin , A drug selected from interferon, immunoglobulin, GM-CSF, G-CSF and glycoprotein. The preparation according to claim 5 or 6, wherein the pH is adjusted to a range of 3 to 4 by mixing a pH adjusting agent with proliposomes. 8. The formulation according to claim 5, 6 or 7, prepared by adding an absorption accelerator selected from fatty acids, fatty acid salts, bile acids, bile salts, salicylic acid or salicylic acid salts as absorption accelerators of the peptide drugs.